This invention pertains to neural networks, and particularly to multinetwork, multielectrode assay devices capable of completely parallel, non-multiplexed recording of action potential signals.
In the last decade, substantial and crucial progress has been made in the validation of primary neuronal cell cultures as pharmacological research platforms. Networks in culture are spontaneously active and produce action potential (AP) patterns that change in a predictable and reproducible manner in response to physical and chemical/pharmacological perturbations of their environment. By growing such networks on arrays of substrate integrated microelectrodes (Gross et al., 1977; Gross 1994), intimate contact with the electrodes can be achieved, providing high signal-to-noise ratios, long-term stability, and an operational lifetime of several months. The monitoring of many nerve cells in such networks yields rich spatio-temporal patterns, subtle sup-population responses, and general fault tolerance, as all data analysis is based on nerve cell groups rather than single neurons. Realistic applications to the fields of toxicology, pharmacology, and drug development are emerging (Gross et al., 1997; Morefield et al., 2000; Gramowski et al., 2000; Keefer et al., 2001a,b, Gross and Gopal, 2006).
It also has been shown that such networks function as biosensors, as they respond to any compound that can alter the normal function of the nervous system and do not require a-priori knowledge (or “fingerprints”) of the agents encountered (Gross et al., 1997, Gross and Pancrazio, 2006). They represent “broad-band” biosensors of interest to the military and homeland security. A stand-alone automated sensor station prototype already exists at the Naval Research Laboratory (NRL/UNT collaboration under DARPA Activities Detection Program). As complex, spontaneously active systems, the networks also embody the mystery of pattern generation, processing, and storage, and therewith, the initial steps and strategies of information processing.
Two-network arrays for simultaneous recording from two separate networks (32 electrodes each) are now in routine use at select institutions. However, this approach is still far from what is needed for efficient exploration and applications of network dynamics. Statistical evaluations of interculture repeatability, multiple drug and sequential concentration applications, and of the temporal evolution of toxic responses are tedious and far too slow for present industrial and even research requirements.
A major temporal and financial burden associated with drug development and toxicology assessment is the lack of physiological assay platforms that are positioned between the biochemist and the animal or human experiments in order to provide rapid, quantitative data on neuroactive/neurotoxic responses. What is lacking in the art is a platform that uses the complex and sensitive neurophysiological network dynamics (provided by the simultaneous recording of action potentials from hundreds of nerve cells), for determination of pharmacological and toxicological responses. By employing many networks in parallel in an automated, robotic system, it would be highly desirable to develop a highly-efficient screening of compounds that would substantially accelerate research in these areas, and drastically reduce the number of animals required by present testing and evaluation procedures. The central nervous system tissue from embryos of one pregnant mouse (usually 10-12) can produce cell pools that allow the seeding of over 1000 networks. This represents an extraordinary efficiency in tissue utilization.